Fungi in acidic fire: A potential source of industrially important enzymes

The microbial life that exists in harsh habitats of low pH possess several unique characteristics, which assign interesting qualities to these microorganisms and enable them to thrive in such a harsh environment. Among microorganisms inhabiting low pH environments, fungi are the second largest reported organisms. These acidophilic fungi are the main source of acid–stable enzymes that could be utilized in many industries including paper, leather making, food and feed industries, where the efficacy of commonly available enzymes is limited by challenges like stability and functional kinetics. The current review discusses the acidophilic fungi with emphasis on their diversity and pH homeostasis mechanisms adopted against low pH environments. In addition, an overview about the acid–stable enzymes obtained from these acidophilic fungi, their main sources and potential applications have also been discussed.     

Improving the quality of industrially important enzymes by directed evolution

Directed evolution is a new process for developing industrially viable biocatalysts. This technique does not require a comprehensive knowledge of the relationships between sequence structure and function of proteins as required by protein engineering. It mimics the process of Darwinian evolution in a test tube combining random mutagenesis and recombination with screening or selection for enzyme variants that have the desired properties. Directed evolution helps in enhancing the enzyme performance both in natural and synthetic environments. This article reviews the process of directed evolution and its application to improve substrate specificity, activity, enantioselectivity and thermal stability.     

Molecular identification of the industrially important strain Ogataea parapolymorpha

The thermotolerant strain 1-IR was isolated as a contaminant microflora of the industrial strain “Red” (the Netherlands) during the study of the baker’s yeast Saccharomyces cerevisiae. The strain was assigned to the species Ogataea parapolymorpha by sequencing the 26S rDNA D1/D2 domain. The strain 1-IR was shown to be capable of efficient glucose and xylose fermentation at an elevated temperature of 45°C. In this respect, the strain 1-IR surpassed the thermotolerant yeasts O. polymorpha CBS 4732, NCYC 495, and O. parapolymorpha DL1. The prospects of using the O. parapolymorpha yeasts as producers of biofuel from lignocellulose wastes of agricultural and woodworking industries is discussed.     

ARBRE: Computational resource to predict pathways towards industrially important aromatic compounds

Synthetic biology and metabolic engineering rely on computational search tools for predictions of novel biosynthetic pathways to industrially important compounds, many of which are derived from aromatic amino acids. Pathway search tools vary in their scope of covered reactions and compounds, as well as in metrics for ranking and evaluation. In this work, we present a new computational resource called ARBRE: Aromatic compounds RetroBiosynthesis Repository and Explorer. It consists of a comprehensive biochemical reaction network centered around aromatic amino acid biosynthesis and a computational toolbox for navigating this network. ARBRE encompasses over 33′000 known and 390′000 novel reactions predicted with generalized enzymatic reactions rules and over 74′000 compounds, of which 19′000 are known to biochemical databases and 55′000 only to PubChem. Over 1′000 molecules that were solely part of the PubChem database before and were previously impossible to integrate into a biochemical network are included into the ARBRE reaction network by assigning enzymatic reactions. ARBRE can be applied for pathway search, enzyme annotation, pathway ranking, visualization, and network expansion around known biochemical pathways and products of lignin degradation to predict valuable compound derivations. In line with the standards of open science, we have made the toolbox freely available to the scientific community on git (https://github.com/EPFL-LCSB/ARBRE) and we provide the web-version at http://lcsb-databases.epfl.ch/arbre/. We envision that ARBRE will provide the community with a new computational resource and comprehensive search tool to predict and rank pathways towards industrially important aromatic compounds.     

Horseradish peroxidase catalyzed degradation of industrially important dyes.

Horseradish peroxidase (HRP) is known to degrade certain recalcitrant organic compounds such as phenol and substituted phenols. Here, for the first time we have shown HRP to be effective in degrading and precipitating industrially important azo dyes. For Remazol blue, the enzyme activity was found to be far better at pH 2.5 than at neutral pH. In addition, Remazol blue acts as a strong competitive inhibitor of HRP at neutral pH. Horseradish peroxidase shows broad substrate specificity toward a variety of azo dyes. Kinetic constants (K(m)(app) and V(max)(app)) for two different dyes have been determined. In addition to providing a systematic analysis of the potential of HRP in degradation of dyes, this study opens up a new area on exploration of commercial dyes as inhibitors of enzymes. 2001 John Wiley & Sons, Inc.     

Developing Bacillus spp. as a cell factory for production of microbial enzymes and industrially important biochemicals in the context of systems and synthetic biology

Increasing concerns over limited petroleum resources and associated environmental problems are motivating the development of efficient cell factories to produce chemicals, fuels, and materials from renewable resources in an environmentally sustainable economical manner. Bacillus spp., the best characterized Gram-positive bacteria, possesses unique advantages as a host for producing microbial enzymes and industrially important biochemicals. With appropriate modifications to heterologous protein expression and metabolic engineering, Bacillus species are favorable industrial candidates for efficiently converting renewable resources to microbial enzymes, fine chemicals, bulk chemicals, and fuels. Here, we summarize the recent advances in developing Bacillus spp. as a cell factory. We review the available genetic tools, engineering strategies, genome sequence, genome-scale structure models, proteome, and secretion pathways, and we list successful examples of enzymes and industrially important biochemicals produced by Bacillus spp. Furthermore, we highlight the limitations and challenges in developing Bacillus spp. as a robust and efficient production host, and we discuss in the context of systems and synthetic biology the emerging opportunities and future research prospects in developing Bacillus spp. as a microbial cell factory.     

Extracellular enzymes in a polyhumic lake: important regulators in detritus processing

Eight extracellular enzymes and their corresponding natural substrates were studied in an acid polyhumic lake. Highest activities were found for phosphatases (100–150 nmol 1⁻¹ h⁻¹), glucosidase (70–120 nmol 1⁻¹ h⁻¹), and aminopeptidases (20–30 nmol l⁻¹ h⁻¹), Significant relationships were found for natural polymeric substrate composition, variation and enzyme activities. Identified carbohydrates and amino acids contributed 1–5% to the DOC pool and are assumed to undergo significant processing by microbial glycosidases and aminopeptidases. Measured enzymes are partially modified in their activity by lake water acidity, temperature and humic matter. Extracellular enzymes are regarded as important regulators in microbial detritus processing and substrate utilization.     

Thermophilic organisms as sources of thermostable enzymes

Thermostable enzymes are receiving considerable attention and there are many industrial applications already. Most thermostable enzymes on the market have, however, been derived from mesophiles. Further research, especially into the possibilities of cloning enzymes from thermophiles into mesophilic hosts, will greatly increase the exploitation of thermophiles in biotechnology.     

New understandings of thermostable and peizostable enzymes

Recent large-scale studies illustrate the importance of electrostatic interactions near the surface of proteins as a major factor in enhancing thermal stability. Mutagenesis studies have also demonstrated the importance of optimized charge interactions on the surface of the protein, which can significantly augment enzyme thermal stability. Directed evolution studies show that increased stability may be obtained by different routes, which may not mimic those used by nature. Despite observations that some of the most thermotolerant organisms grow under conditions of high pressure, little effort has been made to understand the correlation between pressure and temperature stability. One recent study demonstrates that the active-site volume may be important in increasing pressure stability.     

Evolutionary engineering of Saccharomyces cerevisiae for improved industrially important properties

This article reviews evolutionary engineering of Saccharomyces cerevisiae. Following a brief introduction to the 'rational' metabolic engineering approach and its limitations such as extensive genetic and metabolic information requirement on the organism of interest, complexity of cellular physiological responses, and difficulties of cloning in industrial strains, evolutionary engineering is discussed as an alternative, inverse metabolic engineering strategy. Major evolutionary engineering applications with S. cerevisiae are then discussed in two general categories: (1) evolutionary engineering of substrate utilization and product formation and (2) evolutionary engineering of stress resistance. Recent developments in functional genomics methods allow rapid identification of the molecular basis of the desired phenotypes obtained by evolutionary engineering. To conclude, when used alone or in combination with rational metabolic engineering and/or computational methods to study and analyze processes of adaptive evolution, evolutionary engineering is a powerful strategy for improvement in industrially important, complex properties of S. cerevisiae.     

Endophytes from an Australian native plant are a promising source of industrially useful enzymes

Endophytes are microorganisms that live within plant tissues that are potential sources of novel bioactive compounds, including enzymes. We have identified endophytes of the Australian native plant Eremophilia longifolia which were screened for the production of industrially useful enzymes. Seventeen fungal endophytes were isolated from the leaves of E. longifolia and enzyme production was investigated within a range of pH (3.5, 5.5, 7 and 9) and temperatures (9, 25, 37 and 50 °C). Amylase was the most common enzyme encountered with numerous isolates showing production throughout the temperature and pH ranges. Protease production was also seen over the conditions tested but was more dominant at lower pH and temperature. Activity was not observed for other enzymes including ligninase, xylanase and cellobiohydrolase. Enzymes from isolates of Preussia minima, Alternaria sp. and an unclassified fungus, which showed highest activity in screening assays, were investigated further. Enzyme production was verified by zymography and the amylase activity of P. minima was found to be significantly greater than that of Aspergillus oryzae particularly in alkaline conditions and low temperature which are desirable properties for the detergent industry. This work shows that enzymes with potential use in industry can be readily identified in fungal endophytes.     

Production of thermostable amylolytic enzymes by Thermococcus hydrothermalis

A cell extract of Thermococcus hydrothermalis, grown for 6 h, gave -glucosidase activity at 14.9 U/l, degrading oligosaccharides and maltose. -Amylase, -glucosidase and pullulanase activities were detected at 289 U/l, 13.5 U/l and 30 U/l respectively in the culture medium after 24 h growth of the archaeum. All of three enzymes, characterised by a half-life time of 1 to 5 h at 95°C, degraded both the (14) and (16) linkages of polysaccharides and the (14) linkages of oligosaccharides. © Rapid Science Ltd. 1998     

Kinetics of deactivation for thermostable DNA polymerase enzymes

Summary The kinetics of thermal deactivation for thermostable DNA polymerase enzymes were investigated by using the experimental data published elsewhere (Nielson et al. 1996. Strategies. 9, 7–8). The order of deactivation (a) and the deactivation rate constants (k _d) were determined for different Taq DNA polymerase enzymes and were found to be of first order.     

Self-assembled surfactant nano-structures important in drug delivery: a review

Role of self assembled structures as a vehicle is significant over the years. Their applications have been found for all routes of drug delivery. These micro and nano structures are containers loaded with drugs, ideal for targeted and sustained release of the drug. Drug efficacy depends on the drug loaded into the vehicle, temperature, drug solubility, pH, release characteristics, additives and most significantly, the vehicle morphology. This in turn suggests that the same vehicle cannot be used with high efficiency for all types of drugs and locations where the drug delivery has to take place. The status of various self assembled structures and their applications in drug delivery is reviewed in this communication.